1. Field of the Invention
The present invention relates generally to the fields of molecular immunology and immunodiagnosis. More specifically, the present invention relates to a surface protein from Ehrlichia canis, P43, useful as an antigen in the immunodiagnosis of Canine Ehrlichiosis.
2. Description of the Related Art
Canine monocytic ehrlichiosis is a potentially fatal tick-borne disease of dogs with worldwide distribution caused primarily by the rickettsial agent, Ehrlichia canis (8). E. canis is an obligately intracellular bacterium that exhibits tropism for monocytes and macrophages (13), and establishes persistent infections in the vertebrate host (7). The disease is characterized by three stages: the acute stage which lasts 2 to 4 weeks, the subclinical stage, in which dogs can remain persistently infected for years, but do not exhibit clinical signs, followed by the chronic phase, where in many dogs the disease becomes progressively worse due to bone marrow hypoplasia and the prognosis less favorable (19). Treating the disease in the acute phase is important for the best prognosis, but clinical presentation of canine ehrlichiosis is non-specific making diagnosis difficult. Hematologic abnormalities such as leukopenia and thrombocytopenia often provide useful evidence of canine ehrlichiosis and are important factors in the initial diagnosis (19).
Diagnosis of canine ehrlichiosis by serologic methods such as the indirect fluorescent-antibody (IFA) test has become the standard method due to its simplicity, reliability and cost effectiveness (19). However, shortcomings of the indirect fluorescent-antibody test include the inability to make a species-specific diagnosis due to antigenic cross reactivity with other closely related Ehrlichia species that infect dogs (E. chaffeensis, E. ewingii, E. equi, and E. platys), subjective interpretations, which may result in false-negative results, or false-positives caused by cross-reactive antigens. Other diagnostic methods such as polymerase chain reaction (PCR) have been developed for specific detection of E. canis, and were reported to be more sensitive than cell culture isolation, but this method requires specialized training and expensive equipment (9). Isolation of the organism is time consuming, and only a few laboratories have been consistently successful with this method. Furthermore, additional tests characterizing the isolate are required for defining a specific etiology using this method.
Serologically cross-reactive antigens shared between E. canis and E. chaffeensis have been reported. Some of the major serologically cross-reactive proteins exhibit molecular masses of 28-30-kDa (1, 16), and it is now known that these proteins are encoded by homologous multigene families (14, 15). There are 21 and 5 homologous, but nonidentical, p28 genes that have been identified and sequenced in E. chaffeensis and E. canis, respectively (11, 28). Similar intraspecies and interspecies strain homology was observed between the P28 proteins of E. canis and E. chaffeensis, explaining the serologic cross reactivity of these proteins (10). A recent report demonstrated that the rP28 protein from E. chaffeensis was an insensitive tool in diagnosing cases of human monocytotrophic ehrlichiosis (HME) (25). The underlying reason appears to be the variability of the P28 protein among different strains of E. chaffeensis (27). Conversely, the P28 genes identified in E. canis are conserved among geographically dispersed strains (10, 11), and the E. canis rP28 has proven to be useful for diagnosis of canine ehrlichiosis (10, 14). Other homologous immunoreactive proteins including the glycoproteins P140 and P120 in E. canis and E. chaffeensis, respectively, have been cloned (24, 26). Reactivity of the rP120 of E. chaffeensis has correlated well with the IFA for serodiagnosis of human monocytotropic ehrlichiosis, and preliminary studies with the rP140 of E. canis suggest that it may be a sensitive and reliable immunodiagnostic antigen (25, 26).
The prior art is deficient in the lack of Ehrlichia canis-specific antigen for the immunodiagnosis of canine ehrlichiosis. The present invention fulfills this longstanding need and desire in the art.
In this study, a new highly immunoreactive E. canis protein gene of 1170-bp encoding a protein with predicted molecular mass of 42.6-kD was cloned. The gene was not detected in E. chaffeensis DNA, and antibodies against the P43 did not react with E. chaffeensis antigen by IFA. The protein was localized to the surface of E. canis by immunoelectron microscopy. Use of the rP43 protein for serodiagnosis of canine ehrlichiosis was compared to previously described immunoreactive E. canis rP28 and rP140 proteins. E. canis rP43 and rP28 were found to be the sensitive and reliable for the serologic diagnosis of canine monocytotrophic ehrlichiosis.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.
In one embodiment of the current invention, DNA encoding Ehrlichia canis immunoreactive surface protein P43 is described. In addition to the DNA sequence described herein, this isolated DNA may also consist of DNA which hybridizes to the P43 DNA and encodes a P43 protein or DNA encoding a P43 protein but differing in codon sequence due to the degeneracy of the genetic code. Preferably, the DNA has the sequence shown in SEQ ID No: 1 and the P43 protein has the amino acid sequence shown in SEQ ID No: 2.
In another embodiment of the instant invention, a vector is provided comprising the P43 DNA and regulatory elements necessary for the expression of the P43 gene in a cell. This vector may be transfected into host cells selected from bacterial cells, mammalian cells, plant cells or insect cells. The bacterial cells may be E. coli cells.
In a yet another embodiment of the instant invention, an isolated and purified Ehrlichia canis immunoreactive surface protein is provided. This P43 protein may be encoded by the DNA described herein. Alternatively, the protein may be encoded by DNA which hybridizes to the DNA described herein or DNA which differs in nucleotide sequence but encodes the same due to the degeneracy of the genetic code. In a preferred embodiment, the protein has the amino acid sequence shown in SEQ ID No: 2.
In another embodiment of the instant invention, a n antibody may be directed against the P43 protein. In one embodiment, this antibody is a monoclonal antibody.
In yet another embodiment of the instant invention, the P43 protein may be used in the preparation of a vaccine against canine ehrlichiosis.
In a further embodiment of the instant invention, a method of determining whether a dog is infected with Ehrlichia canis is provided by testing whether serum from a potentially infected dog reacts to E. canis P43 protein. The P43 protein used may be a recombinant P43, and western blot analysis may be used to determine whether the dog""s serum reacts to the P43 protein antigen. Since reactivity to P28 is also a reliable marker for Ehrlichia canis infection, reaction to both antigens may be used for a conclusive diagnosis.
In yet another embodiment of the instant invention, serodiagnostic kit is provided for determining whether a dog is infected with Ehrlichia canis. The kit is comprised immobilized Ehrlichia canis antigens (P43, P28 or both), appropriate dilution buffers for dog serum, anti-dog serum second antibody linked to a reporter molecule, appropriate reagents for detection of said reporter molecule. The antigens may be immobilized on membranes or linked to microtiter plates. The reporter molecule may be luciferase, alkaline phosphotase, horseradish peroxidase, xcex2-galactosidase, or a fluorescent label.
In another embodiment of the instant invention, a PCR amplification method is provided for whether a dog has been infected with Ehrlichia canis. DNA is extracted from the blood of a potentially infected dog and subjected to PCR amplification with oligonucleotide primers specific for the E. canis P43 gene. The resulting PCR amplification products are separated by size by a method such as gel electrophoresis and detection of an appropriately sized product indicates Ehrlichia canis infection. Examples of appropriate oligonucleotide primers are SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, and SEQ ID No. 12.
In yet another embodiment of the instant invention, a kit is provided for PCR detection of the P43 gene in dog blood. The kit comprises reagents for DNA extraction from blood, P43-specific oligonucleotides, and reagents for PCR amplification.